The present invention relates to a method and apparatus for the detection of Green Fluorescence Protein GFP).
GFP is found in the jellyfish Aequorea victoria. With the ability to clone and express GFP in a diverse range of cells and organisms including bacteria, yeast, plants and higher animals, GFP has become a versatile fluorescent marker for monitoring physiological processes, visualising protein localisation and detecting the expression of transferred genes [Green Fluorescent Proteins, Proteins, Properties, Applications and Protocols, ed. M. Chalfie and S. Kain, Wiley and Sons, 1st edn., 1998; H-H Gerdes and C. Kaether, FEBS Lett., 1996, 389, 44; A. B. Cubitt, R. Heim, S. R. Adams, A. E. Boyd, L. A. Gross and R. Y. Tsien, Trends Biol. Sci., 1995, 20, 448]. The usefulness of GFP stems from the fact that fluorescence from GFP requires no additional co-factors; the fluorophore is self-assembling via a cyclization reaction of the peptide backbone.
GFP is bio-compatible, and when used as a tag does not alter the normal function or localisation of a protein to which it is fused. Proteins, cells and organelles marked with GFP can be visualised and monitored in living tissue without the need for fixation. Hence the dynamics of cellular processes can be non-invasively quantified in real time using GFP, simply by the measurement of fluorescence.
The wild-type GFP consists of 238 amino acids and has a cylindrical structure with the fluorophore element encapsulated in the centre [F. Yang, L. G. Moss and G. N. Phillips, Jr., Nat. Biotechol., 1996, 14, 1246]. As such it is a very chemically and photochemically stable and resilient fluorophore. Bright green fluorescence at 508-515 nm is readily induced by illumination of GFP with visible blue light at 470 nm. Genetic modification of GFP has been used to provide several useful mutants with fluorescence that is significantly blue or yellow shifted.
One application of GFP is in the development of an automated flow-injection bioassay for the detection of genotoxic compounds and the quantification of genotoxicity. The basis of the method is the use of yeast cells that are genetically modified such that they produce GFP in response to the activation of the cells"" DNA repair mechanisms by DNA damage. The presence, concentration or potency, of a suspected genotoxic compound can be quantified by measuring an increase in green fluorescence from intact yeast cells [R. M. Walmsley, N. Billinton and W.-D. Heyer, Yeast, 1997, 13, 1535; N. Billinton, M. G. Barker, C. E. Michael, A. W. Knight, N. J. Goddard, P. R. Fielden and R. M. Walmsley, Biosens. Bioelectron., 1998, 13, 831; A. W. Knight, N. J. Goddard, P. R. Fielden, M. G. Barker, N. Billinton and R. M. Walmsley, Meas. Sci. Technol., 1999, 10, 211].
Organs or organelles within organisms or cells where GFP is localised can often be readily visualised and distinguished from the background matrix by fluorescence microscopy techniques. However, in cases where GFP is only weakly expressed, or where GFP is in free solution such as in the cell cytosol, the fluorescence signal from GFP is invariably contaminated by cellular or media auto-fluorescence [K. D. Niswender, S. M. Blackman, L. Rohde, M. A. Magnuson and D. W. Piston, J. Microsc., 1995, 180, 109]. This has also been the case in yeast cell studies. In an analytical context this restricts the lower limit of detectable signal.
The term auto-fluorescence refers to fluorescence arising from any species other than GFP, known or unknown, naturally occurring or added, which is significantly bright at the wavelength of GFP fluorescence.
Green auto-fluorescence is almost universal to all living cells and organisms, and arises from a diverse range of sources. Likely chemical sources are reduced nicotinamide dinucleotides, oxidised flavins, age-related pigments and oxidised aromatic amino acids such as tryptophan. However, in many cases the exact source of auto-fluorescence is unknown. In general the brightness of the auto-fluorescence increases with the age of the cell or organism. Lipofuscins is a general term name for the auto-fluorescence that accumulates in (particularly ageing) mammalian cells. Auto-fluorescence has been noted to cause difficulties in the quantification of GFP expressed in many cells and species, for example:
Mammalian Heart Cells (Auto-fluorescence arising from myocardium); T. Kawada, W. S. Shin, Y. Nakatsuru, T. Koizumi, A. Sakamoto, T. Nakajima, Y. OkaiMatsuo, M. Nakazawa, H. Sato, T. Ishikawa, T. ToyoOka. Precise identification of gene products in hearts after in vivo gene transfection, using sendai virus-coated proteoliposomes. Biochemical and Biophysical Research Communication, 1999, 259, 408.
Plants (Auto-fluorescence arising from cell walls in maize plants); A. H. M. vanderGeest, J. F. Petolino. Expression of a modified green fluorescent protein gene in transgenic maize plants and progeny. Plant Cell Reports, 1998, 17, 760.
Nematodes (Roundworms) (Auto-fluorescence mainly from the gut); S. Hashmi, M. A. AbuHatab, R. R. Gaugler. Green fluorescent protein a versatile gene marker for entomopathogenic nematodes, Fundamental and Applied Nematology, 1997, 20, 323; Green Fluorescent Proteins: Proteins, Properties, Applications and Protocols, ed. M. Chalfie and S. Kain, Wiley and Sons, 1st edn., 1998, p. 154.
Drosophila (Fruit flies); J. D. Plautz, R. N. Day, G. M. Dailey, S. B. Welsh, J. C. Hall, S. Halpain, S. A. Kay. Green fluorescent protein and its derivatives as versatile markers for gene expression in living Drosophila melanogaster, plant and mammalian cells. Gene, 1996, 173, 83; Green Fluorescent Proteins: Proteins, Properties, Applications and Protocols, ed. M. Chalfie and S. Kain, Wiley and Sons, 1st edn., 1998, p. 172.
Bacteria; P. J. Lewis, J. Errington. Use of green fluorescent protein for detection of cell-specific gene expression and subcellular protein localization during sporulation in Bacillus subtilis. Microbiology-UK, 1996, 142, 733.
Yeast; Green Fluorescent Proteins: Proteins, Properties, Applications and Protocols, ed. M. Chalfie and S. Kain, Wiley and Sons, 1st edn., 1998, p. 149.
Many methods have been used to reduce the effect of auto-fluorescence upon GFP measurements, with varying degrees of success. However, each of the known methods suffers from disadvantages.
A first known method of reducing the effect of auto-fluorescence upon GFP measurements involves designing an optimised set of optical narrow-band filters to specifically pick out regions of the optical spectrum where GFP can be both excited, and emit light, with greater efficiency than the auto-fluorescence (see for example M. J. Zylka and B. J. Schnapp. Optimized filter set and viewing conditions for S65T mutant of GFP is living cells. Biotechniques, 1996, 21, 220). In most applications, the standard optical filter sets for fluorescein are not specific enough to discriminate GFP from auto-fluorescence. Dedicated excitation and emission filter sets for GFP have recently been made commercially available, although they are not suitable for all existing instrumentation. These filter sets suffer from the disadvantage that they are currently relatively expensive at several hundred pounds sterling per set. A further disadvantage is that in many cases the excitation and emission spectra of the auto-fluorescence significantly overlap those of GFP, making it difficult to distinguish between GFP fluorescence and auto-fluorescence signals.
Most researchers are required to optimise their own set of filters, using filters from various commercial sources, for their own particular applications. This is for two main reasons. Firstly, auto-fluorescence arises from a disparate, and often unknown range of chemicals, and varies enormously between different cells and species, and even over the lifetime of an individual cell. Secondly, new GFP mutants with diverse spectroscopic properties are becoming available all the time.
It should be noted the greatest wavelength discrimination achievable using optical narrow-band filters is by the use of multiple overlapping filters. However, where this is done a significant proportion of the available fluorescence signal is lost with each filter, (typically 30 to 50%), hence reducing measurement sensitivity.
Enhanced measurement of GFP can be made under a microscope by selectively focussing on one small, defined area of a specimen without illuminating the whole sample. This can be achieved in three dimensions using confocal fluorescence microscopy (see for example G. Jung, J. Wiehler, W. Gxc3x6hde, J. Tittel, Th. Baschxc3xa9, B. Steipe, C. Brxc3xa4uchle. Confocal microscopy of single molecules of the green fluorescent protein. Bioimaging, 1998, 6, 54; K. D. Niswender, S. M. Blackman, L. Rohde, M. A. Magnuson, D. W. Piston. Quantitative imaging of green fluorescent protein in cultured cells: comparison of microscopic techniques, use in fusion proteins and detection limits. Journal of Microscopy, 1995, 180, 109). In a typical arrangement, laser light is reflected by a dichroic mirror and focussed onto a sample by a microscope objective. The emitted fluorescence is collected by the same objective, passes through the dichroic mirror and is spatially filtered from background light by a pinhole (typically a few 100 xcexcm in diameter) located in front of the detector. This approach is useful where auto-fluorescence arises from a distinct subcellular component, such as a cell wall.
A disadvantage of this approach is the requirement for complex and expensive instrumentation. Furthermore, confocal fluorescence microscopy will not completely eliminate auto-fluorescence when that auto-fluorescence is dispersed throughout the sample, or when that auto-fluorescence operates at the same wavelength as GFP. Confocal fluorescence microscopy is most suited for the discrimination of GFP from auto-fluorescence sources which are located away from the focal spot of a microscope.
In a further method of reducing auto-fluorescence, known as two photon excitation spectroscopy, short pulses of near infra-red laser light (typically 100 femto seconds at 100 MHz) are focused onto a sample in the same manner as described above in relation to confocal microscopy (see for example K. D. Niswender et al. (as above); S. M. Potter, C.-M. Wang, P. A. Garrity, S. E. Fraser. Intravital imaging of green fluorescent protein using two-photon laser-scanning microscopy. Gene, 1996, 173, 25). Two-photon excitation occurs on simultaneous absorption of two separate photons, each having half the energy required to cause transition to the excited state. (For example 780 nm light is used to excite a transition that would be excited by light at 390 nm).
Excitation only occurs within a diffraction, limited focal spot, with virtually no fluorescence excitation above or below the focal plane. Hence, a confocal pinhole is not required for spatial resolution and a greater amount of light can be collected than is collected using confugal fluorescence microscopy, thus enhancing the sensitivity of the technique. Any reflected infra-red excitation light is easily removed from the fluorescence emission signal, since it is separated in wavelength from the fluorescence.
Two photon excitation spectroscopy has several disadvantages. The first is the need for complex and expansive instrumentation (pulsed lasers, etc.). The second is that common wavelengths from infra-red lasers tend produce excitation at wavelengths below 490 nm, where the effect of auto-fluorescence increases due to efficient excitation of NADH (which is abundant in cells). A further disadvantage of two photon excitation spectroscopy is the rapid photobleaching of GFP.
Time resolved fluorescence spectroscopy (TRFS) and fluorescence lifetime imaging microscopy (FLIM), are often used to quantify or visualise fluorescent labels in the presence of background non-specific auto-fluorescence. This approach is successful, provided that the fluorescence lifetime of a label is significantly longer than that of the auto-fluorescence (see for example R. Pepperkok, A. Squire, S. Geley, P. I. H. Bastiaens. Simultaneous detection of multiple green fluorescent proteins in live cells by fluorescence lifetime imaging microscopy. Current Biology, 1999, 9, 269). A pulsed light source is used, and fluorescence of the label is measured a set time after the short-lived auto-fluorescence has decayed. Common labels are based on osmium, ruthenium or lanthanide chelates, with fluorescence lifetimes typically 20 ns or more.
Since GFP has a particularly short fluorescence lifetime (approximately 2.8 ns) this approach is not particularly useful in the discrimination of GFP and auto-fluorescence. In addition, the method requires complex and expansive instrumentation. FLIM has however been used to distinguish different GFP""s in a multiple labelling application, using the diversity in individual GFP mutant""s fluorescence lifetimes.
Auto-fluorescence in microscopy images can sometimes be corrected for, either visually or by computer picture manipulations given sufficient, separation between the GFP fluorescence wavelength and the auto-fluorescence wavelength. For example, auto-fluorescence is often more yellow-green, rather than pure bright green in appearance. However, such transformations are subjective and inevitably involve corruption of an original image. Correction of this type requires the use of expensive colour detectors.
A further know approach to reducing media auto-fluorescence has been to irradiate a medium with short wavelength ultra-violet light before use (see for example M. J. Zylka et al., full reference given above). This photo-bleaches some components likely to cause autofluorescence. This approach suffers from the disadvantage that it may also destroy media components which are critical for sustaining cells, or may form photochemical derivatives harmful to cells. Culture media can be switched for UV-treated media just prior to measurement, although this necessitates an extra laborious step, and is not applicable to continuous real-time monitoring.
Some researchers have used genetics to solve specific auto-fluorescence problems, by restricting their selection of GFP mutants to those which have emission wavelengths significantly separated from those of the interfering autofluorescence, or which are exceptionally bright (see for example R. H. Kohler, W. R. Zipfel, W. W. Webb, M. R. Hanson.
The green fluorescent protein as a marker to visualize plant mitochondria in vivo. Plant Journal, 1997, 11, 613). In addition, very strong promoters are used such that the chosen GFP is highly expressed, or localised within parts of a cell. This is often used in applications where there is intense auto-fluorescence, such as in green plant tissues. In applications where GFP is not localised or only weakly expressed, the presence of natural auto-fluorescence severely restricts the usefulness of GFP with conventional fluorescence detection.
Many common growth media are highly auto-fluorescent. Researchers have therefore tried to reduce auto-fluorescence from this source by the use of selected minimal, defined media, notably with low concentrations of riboflavin and tryptophan. Such media suffer from the disadvantage that they often prove sub-optimum for cell growth and development compared to richer, more complex, media.
The commonly used S65T GFP mutant is fluorescent from pH 7 to 11.5, whereas some cellular auto-fluorescence is quenched at high pH. Hence it sometimes possible to increase pH (typically to pH 10-11) to reduce background auto-fluorescence (see for example R. M. Walmsley, N. Billinton, W.-D., Heyer. Green fluorescent protein as a reporter for the DNA damage-induced gene RAD54 in Saccharomyces cerevisiae. Yeast, 1997, 13, 1535). However, this is an invasive step which will be detrimental to living cells and is best applied to cellular extracts.
Capillary electrophoresis (CE) has been used to directly separate GFP from other auto-fluorescent proteins and intracellular components (see for example A. Malek, M. G. Khaledi. Expression and analysis of green fluorescent proteins in human embryonic kidney cells by capillary electrophoresis. Analytical Biochemistry, 1999, 268, 262; G. M. Korf, J. P. Landers, D. J. O""Kane. Capillary electrophoresis with laser-induced fluorescence detection for the analysis of free and immune-complexed green fluorescent protein. Analytical Biochemistry, 1997, 251, 210). CE is widely used in biomedical applications due to the small sample volumes required and rapid analysis time. CE is a high resolution separation technique in which molecules to be separated migrate in an electric field, with mobilites that depend on molecular size and charge. Separation takes place in a capillary tube across which a voltage of 10""s of kV is applied. In the case of GFP, CE is coupled with laser fluorescence detection.
The technique works best for cellular extracts, however these must be laboriously prepared by washing and lysis of the cells, followed by centrifugation, filtration and re-suspension steps. Using CE it is possible to separate different forms of GFP, and bound from unbound GFP for example using antibody labels. Whole cells may be analysed, but only for separation from auto-fluorescent components arising from the growth medium.
CE is inherently destructive and requires expensive instrumentation (high voltage power supply, laser induced fluorescence detector). It can also be difficult to optimise the CE buffer conditions such as pH, to suit both the separation and GFP fluorescence, whilst in some cases also sustaining cells.
It is apparent from the above that the problem of how to reduce the effect of auto-fluorescence upon GFP measurements is well recognised, and that a wide variety of methods have been developed in order to try and minimise the effect of auto-fluorescence. All of the known methods suffer from significant disadvantages.
It is an object of the present invention to provide a method and apparatus for discriminating GFP and auto-fluorescence which overcomes at least some of the above disadvantages.
According to a first aspect of the invention, there is provided a method of distinguishing between fluorescent light emitted by green fluorescent protein and fluorescent light emitted by auto-fluorescent molecules, the method comprising:
using plant-polarised light to illuminate a sample containing green fluorescent protein and auto-fluorescent molecules;
detecting the intensity of fluorescent light that is emitted with a first polarisation from the sample;
detecting the intensity of fluorescent light that is emitted with a second polarisation from the sample; and
subtracting a first of said detected intensities from a second of said detected intensities to obtain a difference signal.
The inventors have realised that a sample, when illuminated by polarised light, provides strongly polarised GFP fluorescence and significantly less polarised auto-fluorescence. The difference signal obtained according to the invention thus distinguishes between light emitted by GFP and light emitted by auto-fluorescent molecules. The invention provides a new and straightforward method for distinguishing GFP fluorescence from auto-fluorescence.
The term auto-fluorescent molecule is intended to include any molecule other than GFP, known or unknown, naturally occurring or added, which is significantly bright at the wavelength of GFP fluorescence.
A polarised light source may either produce polarised light directly, for example an argon ion laser, or may produce unpolarised light which is polarised upon passing through a polarising filter placed between the light source and the sample. Examples of suitable light sources include lasers, LED""s, xenon lamps, mercury lamps and halogen lamps.
Any suitable detector may be used to detect the fluorescent light, For example, a photomultiplier tube detector, a silicon photodiode, a CCD camera, video camera, photographic film and the eye. One or more polarising filters may be used to select a particular polarisation of fluorescent light for detection.
The inventors have determined that for GFP in free solution, or contained within a cytosol of a yeast cell, the intensity of fluorescence perpendicularly polarised with respect to a polarised light source (Il) is approximately 50% less than the intensity of fluorescence aligned parallel to the polarised light source Ill).
Measured fluorescence polarisation (P) may be defined as follows:
P=(Ill-Il)/(Il+Il) 
The difference between Ill and Il is large for GFP fluorescence, since the GFP fluorescence is significantly polarised, but much less for background auto-fluorescence. Thus by measuring the difference signal (Ill-Il) it is possible to discriminate between GFP and auto-fluorescence, even when the GFP and auto-fluorescence occur at the same wavelength.
The inventors believe that the strong polarisation of GFP fluorescence arises as a result of the large size, and hence slow rotation rate, of GFP in solution. When fluorescent molecules are illuminated by plane polarised light, those molecules with an electronic transition moment aligned parallel to the electric vector of the excitation light are excited. Since GFP is a relatively large molecule and the fluorophore element of GFP is rigidly encapsulated within a cylindrical structure, the fluorophore element of GFP rotates at a rate which is slow compared to the rate at which it fluoresces (the fluorescence lifetime of GFP is approximately 2.8 ns [A. W. Knight, N. J. Goddard, P. R. Fielden, M. G. Barker, N. Billinton and R. M. Walmsley. Anal. Commun., 1999, 36, 113; B. P. Cormack, G. Bertram, M. Egerton, N. A. R. Gow, S. Flakow and A. J. P. Brown, Microbiology, 1997, 143, 303]). Consequently, GFP fluorescence is strongly polarised parallel to the plane of polarisation of the illumination. In contrast to GFP, the molecules that give rise to auto-fluorescence are commonly smaller. Fluorescence emissions from such molecules are largely free of polarisation, since the molecules are free to rotate during the time taken for the electronic transitions of fluorescence emission to occur. In other words, the rotational relaxation time of the molecules is much shorter than the fluorescence decay time and the molecular orientation effectively becomes randomised before fluorescence occurs.
The method according to the first aspect of the invention will distinguish between fluorescent light emitted by green fluorescent protein and fluorescent light emitted by auto-fluorescent molecules, provided that the auto-fluorescent molecules emit fluorescent light that is less polarised than fluorescent light emitted by the green fluorescent protein (i.e. provided that the auto-fluorescent molecules emit fluorescent light with a lower polarisation ratio).
The inventors are aware of a paper that suggests that GFP can be used to replace fluorescein in many biochemical analyses [S. H. Park, R. T. Raines. Green fluorescent protein as a signal for protein-protein interactions. Protein Science, 1997, 6, 2344]. The paper describes monitoring a change of the polarisation of GFP fluorescence when GFP binds to an S-protein fragment of ribonuclease A. This change of polarisation is used to probe protein-protein interactions and obtain binding coefficients. However, although the method described in the paper measures relative values of polarisation of GFP fluorescence, the method does not measure the absolute strength of polarisation of the GFP fluorescence. The paper does not address or refer to the absolute strength of the polarisation itself, and furthermore does not address or refer to the strength of the polarisation of background auto-fluorescence. Thus, there is nothing in this paper to indicate or suggest that GFP fluorescence and background auto-fluorescence have significantly different strengths of polarisation, or to indicate or suggest that a polarisation measurement may be used to distinguish between GFP fluorescence and background auto-fluorescence.
The inventors are aware of a paper that mentions steady-state fluorescence anisotropy of GFP [K. D. Niswender, S. M. Blackman, L. Rohde, M. A. Magnuson, D. W. Piston. Quantitative imaging of green fluorescent protein in cultured cells: comparison of microscopic techniques, use in fusion proteins and detection limits. Journal of Microscopy, 1995, 180, 109]. This paper mentions that GFP fused with glutathione-S-transferase shows a fluorescence anisotropy of 0.29 and 0.34 when excited with light at 395 and 490 nm respectively. The paper mentions the different anisotropy values to suggest that different excited states and decay pathways may be involved depending on the wavelength of excitation. It is suggested that this may explain why photobleaching rates appear to be dependent on excitation wavelength. The paper does not address or refer to the strength of the polarisation of the GFP fluorescence, and furthermore does not address or refer to the strength of the polarisation of background auto-fluorescence. There is nothing in this paper to indicate or suggest that GFP fluorescence and auto-fluorescence have significantly different strengths of polarisation, or to indicate or suggest that a polarisation measurement may be used to distinguish between GFP fluorescence and auto-fluorescence. This is despite the fact that the paper is addressed towards the use of optical methods to combat auto-fluorescence.
The method according to the first aspect of the invention may be used in conjunction with any instrumentation, whether specially constructed for fluorescence polarisation measurement or adapted by the insertion of polarising filters. The method may be applied to flow through instrumentation (as described in detail below) or used with a conventional fluorimeter, a microscope, or a simple camera or CCD detector with a polarising filter placed between the illuminated sample and detector. The method may be used for the bulk analysis of solutions, or for visualisation applications of cells and organisms.
The method according to the first aspect of the invention is advantageous when compared to the prior art for several reasons. The method does not require expensive optimised filter sets, but instead uses inexpensive, easily modified polarising filters, and standard wider band filters. The method does not require complex and expensive instrumentation, as is required for two photon excitation spectroscopy, or for time resolved fluorescent spectroscopy. Where purification of a sample is carried out, as described above, the invention may allow a simpler, faster, less expensive purification method to be used, resulting in a xe2x80x98dirtierxe2x80x99 sample from which GFP fluorescence may be detected.
In some applications of the first aspect of the invention it may be preferable to make a measurement of xe2x80x98brightnessxe2x80x99, rather than to simply measure fluorescence intensity. For example, GFP in yeast cells may be measured using the invention. Yeast cells will grow throughout the duration of an experiment, and a form of normalisation is required to convert the fluorescence detected during the experiment to a xe2x80x9cbrightness per cellxe2x80x9d measurement. The brightness is calculated as: brightness=(Ill-Il)/Cell density measurement, where cell density is quantified by either a turbidimetric or nephelometric method, or any other suitable cell counting method.
WO 98/44149 discloses methods of detecting the presence of an agent that causes or potentiates DNA damage, which involves subjecting cells to a putative DNA damaging agent and monitoring the expression of a light emitting reporter protein, such as GFP, from the cells.
WO 98/44149 further discloses recombinant DNA molecules comprising a regulatory element, which activates gene expression in response to DNA damage, that is operatively linked to a DNA sequence that encodes GFP or a derivative thereof. It also discloses recombinant vectors containing such DNA molecules. Each of these molecules and vectors may be used to transform cells (e.g. yeast) and may be used according to the DNA detecting method described in WO 98/44149.
The inventors have found that the methods described in WO 98/44149 (which are incorporated herein by reference) may be adapted, and improved, such that light emitted from GFP in response to DNA damage may be distinguished from auto-fluorescent molecules according to the method of the present invention. This allows the detection of DNA damage with improved sensitivity. Thus according to a preferred embodiment of the present invention light emitted from GFP contained within or derived from host cells genetically engineered to express GFP (e.g. yeast genetically engineered to express GFP in response to DNA damage) may be distinguished from auto-fluorescence and thereby provide an improved manner of monitoring for GFP expressed from said cells.
The method according to the first aspect of the invention is therefore useful for improving the sensitivity of various biological assays that rely upon light emitted from GFP as a marker. It will be appreciated that recombinant DNA technology may be utilised to allow expression of GFP to be regulated by many different signals (e.g. DNA damage) and therefore the utility of GFP as a marker, and thereby the benefits of the method of the present invention will be wide ranging.
The method according to the first aspect of the invention may also be applied to apparatus that produces an image, wherein two images taken of fluorescent light having two different polarisations are digitally or otherwise subtracted. Areas of the image arising from auto-fluorescence should be reduced in intensity to a much greater extent than those areas showing GFP fluorescence.
According to a second aspect of the invention there is provided an apparatus for distinguishing between fluorescent light emitted by green fluorescent protein and fluorescent light emitted by auto-fluorescent molecules, the apparatus comprising:
illumination means for illuminating a sample containing green fluorescent protein and auto-fluorescent molecules using plane-polarised light;
detector means for detecting the intensity of fluorescent light that is emitted with a first polarisation from the sample;
detector means for detecting the intensity of fluorescent light that is emitted with a second polarisation from the sample; and
subtraction means for subtracting a first of said detected intensities from a second of said detected intensities to provide a difference signal.